Gene silencing by the RNA interference (RNAi) machinery is an evolutionarily conserved process that is critical for control of gene expression in organisms from yeast to human. Targets of RNAi are recognized through complementary interactions with small RNAs that act as guides in the silencing process. Several discrete classes, encompassing thousands of small RNAs have been identified in recent years. For example, microRNAs interact with members of the ubiquitously expressed Argonaute protein family and together these regulate gene expression networks that impact diverse biological processes. Members of the other branch of the Argonaute family, the Piwi proteins, are expressed specifically in germ cells with mutant animals showing severe defects in gametogenesis that lead to sterility. Until recently, the small RNA partners of Piwi proteins were unknown, and the molecular functions of Piwis in gametogenesis remain so. Recently we discovered the small RNA partners of Piwi proteins in mammals and comprehensively analyzed Piwi interacting RNA (piRNA) in Drosophila. In this proposal I present a focused strategy for analyzing piRNA biogenesis and function in the germline. First, I will characterize small RNAs associated with Piwi proteins during different stages of germ cell development. Second, I will probe the mechanism of piRNA biogenesis in vivo. Finally, I will determine the mechanism of piRNA-Piwi mediated silencing. These studies will produce fundamental insights into the functions of Piwi proteins and piRNAs in germline development, which can be applied for discovery of both diagnostic markers and therapeutic targets of human infertility. Furthermore, if successful, the proposed research has the potential to expand the application of RNAi methods by providing tools to manipulate the epigenetic structure of specific loci in mammalian germ cells. [unreadable] [unreadable] [unreadable]